Volume 67, Issue 5, Pages 1844-1854 (May 2005) Ouabain-induced endocytosis of the plasmalemmal Na/K-ATPase in LLC-PK1 cells requires caveolin-1  Jiang Liu, Man Liang, Lijun Liu, Deepak Malhotra, Zijian Xie, Joseph I. Shapiro  Kidney International  Volume 67, Issue 5, Pages 1844-1854 (May 2005) DOI: 10.1111/j.1523-1755.2005.00283.x Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 1 Depletion of cholesterol prevents ouabain-induced early endosomal accumulation of the Na/K-ATPase, EGFR, and c-Src in response to ouabain, and cholesterol repletion restores the control pattern. (A) Shown are representative Western blots. Ten μg of total protein was applied to each lane. (B) Shown is quantification of N = 5 ouabain treated early endosome fractions compared with N = 5 control early endosome fractions (values averaged and expressed as fraction of control). O, ouabain; MCD, methyl β-cyclodextrin; R, cholesterol repletion. Data shown as mean ± SEM. *P < 0.01 vs. control, #P < 0.01 vs. MCD + O. Kidney International 2005 67, 1844-1854DOI: (10.1111/j.1523-1755.2005.00283.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 Caveolin-1 expression in P-11 and C2-9 cells (A) and ouabain-sensitive 86Rb+-uptake assay in P-11 and C2-9 cells (B, expressed as% control). In (A), 20 μg of whole cell lysate was applied to each lane and immunoblotted for caveolin-1. In (B), both cells were treated with or without (as control) ouabain (50 nmol/L) for different amounts of time. N = 4 experiments are represented at each data point in each panel. **P < 0.01 vs. control. (C) Confocal immunofluorescence images of control (upper panel) and ouabain (50 nmol/L for 12 hours on basolateral aspect, lower panel) treated P-11 cells grown to monolayer. In these images, α1 subunit of Na/K-ATPase is labeled with Alexa Fluor® 488, and caveolin-1 is labeled with Alexa Fluor® 546-conjugated secondary antibody. (D) Same conditions as (C), but in C2-9 cells. Size bar = 10 μm. Kidney International 2005 67, 1844-1854DOI: (10.1111/j.1523-1755.2005.00283.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 2 Caveolin-1 expression in P-11 and C2-9 cells (A) and ouabain-sensitive 86Rb+-uptake assay in P-11 and C2-9 cells (B, expressed as% control). In (A), 20 μg of whole cell lysate was applied to each lane and immunoblotted for caveolin-1. In (B), both cells were treated with or without (as control) ouabain (50 nmol/L) for different amounts of time. N = 4 experiments are represented at each data point in each panel. **P < 0.01 vs. control. (C) Confocal immunofluorescence images of control (upper panel) and ouabain (50 nmol/L for 12 hours on basolateral aspect, lower panel) treated P-11 cells grown to monolayer. In these images, α1 subunit of Na/K-ATPase is labeled with Alexa Fluor® 488, and caveolin-1 is labeled with Alexa Fluor® 546-conjugated secondary antibody. (D) Same conditions as (C), but in C2-9 cells. Size bar = 10 μm. Kidney International 2005 67, 1844-1854DOI: (10.1111/j.1523-1755.2005.00283.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 3 Depletion of caveolin-1 prevents ouabain-induced decreases of the plasmalemmal Na/K-ATPase α-1 subunit. (A) Shown is the biotinylated surface Na/K-ATPase α-1 subunit in P-11 and C2-9 cells. After treatment with or without (as control) ouabain (50 nmol/L, 12 hours), cell surface proteins were biotinylated, pull-down with streptavidin, and immunoblotted with the Na/K-ATPase α-1 subunit. O, ouabain. N = 4 experiments are represented at each data point in each panel, **P < 0.01 vs. control. (B) shows quantification data of (A). Kidney International 2005 67, 1844-1854DOI: (10.1111/j.1523-1755.2005.00283.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 4 Depletion of caveolin-1 prevents ouabain-induced increases in nuclear accumulation of biotinylated α-1 subunit of Na/K-ATPase. (A) Shown is representative Western blot following streptavidin isolation of biotinylated α-1 subunit from P-11 and C2-9 cell nuclei from cells exposed to ouabain for 12 hours and control cells. Twenty μg of total protein from nuclear fraction was applied to each lane and immunoblotted with antibody against α-1. (B) Shown is quantification of the nuclear biotinylated pump (data shown as mean ± SEM of 4 individual experiments normalized to the mean control value) in response to ouabain. **P < 0.01 vs. control. Kidney International 2005 67, 1844-1854DOI: (10.1111/j.1523-1755.2005.00283.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 5 Depletion of caveolin-1 prevents ouabain-induced endocytosis of the Na/K-ATPase α-1 subunit. (A) Representative Western blot illustrating effect of ouabain (1 hour at 50 nmol/L) on CCV α-1 subunit of Na/K-ATPase protein content. Five μg of protein was applied to each lane. Quantification of 4 experiments showed that, in P-11 cells, the relative CCV Na/K-ATPase α-1 subunit content in ouabain-treated cells was 2.42 ± 0.45 (P < 0.01 vs. control) compared with CCVs isolated from untreated cells; there is no significant change in C2-9 cells. (B) Quantification of data of (A). (C) Representative Western blot illustrating effect of ouabain (O) at 50 nmol/L for 2 hours on endosomal α-1 subunit of Na/K-ATPase protein content. Ten μg of protein were applied to each lane. (D) Quantification of data of (C). For both (B) and (C), N = 4, data shown as mean ± SEM. *P < 0.05, **P < 0.01 vs. control, #P < 0.01 vs. P-11+ouabain. Kidney International 2005 67, 1844-1854DOI: (10.1111/j.1523-1755.2005.00283.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 6 Depletion of caveolin-1 abolishes ouabain (O)-stimulated protein-protein interaction between the Na/K-ATPase α-1 subunit, PI(3)K p85α subunit, CHC, and AP-2. Coimmunoprecipitate experiments were performed as described before6. One mg of total protein was used for each sample. N = 4, **P < 0.01 vs. control. Kidney International 2005 67, 1844-1854DOI: (10.1111/j.1523-1755.2005.00283.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 7 Depletion of caveolin-1 prevents ouabain-induced compartmentalization of signaling molecules in endosomes. Both P-11 and C2-9 cells were treated with (50 nmol/L, 2 hours) or without (as control) ouabain. (A) A representative Western blot (for EGFR, c-Src, and total ERK) from early endosomal isolation. Ten μg of total protein was applied to each lane, and immunoblotted with antibodies against EGFR, c-Src, and total ERK. (B) Quantitative data expressed as fraction of control (control values = 1). N = 4 control and N = 4 ouabain-treated cells studied. **P < 0.01 vs. control. P-11 + O and C2-9 + O were compared to P-11 and C2-9 control cells, respectively. O, ouabain. Kidney International 2005 67, 1844-1854DOI: (10.1111/j.1523-1755.2005.00283.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 8 EGF-induced activation of EGFR is not sufficient to induce Na/K-ATPase internalization. Both P-11 and C2-9 cells were treated with (50 ng/mL, 15 minutes) or without (as control) EGF. (A) A representative Western blot from early endosomal fractions. Ten μg of total protein was applied to each lane and immunoblotted with antibodies against EGFR, c-Src, total ERK, and α-1 subunit. (B) Quantitative data expressed as fraction of control (control values = 1). N = 3 controls and N = 3 ouabain-treated cells studied. **P < 0.01 vs. control. P-11 + EGF and C2-9 + EGF were compared to P-11 and C2-9 control cells, respectively. Kidney International 2005 67, 1844-1854DOI: (10.1111/j.1523-1755.2005.00283.x) Copyright © 2005 International Society of Nephrology Terms and Conditions

Figure 8 EGF-induced activation of EGFR is not sufficient to induce Na/K-ATPase internalization. Both P-11 and C2-9 cells were treated with (50 ng/mL, 15 minutes) or without (as control) EGF. (A) A representative Western blot from early endosomal fractions. Ten μg of total protein was applied to each lane and immunoblotted with antibodies against EGFR, c-Src, total ERK, and α-1 subunit. (B) Quantitative data expressed as fraction of control (control values = 1). N = 3 controls and N = 3 ouabain-treated cells studied. **P < 0.01 vs. control. P-11 + EGF and C2-9 + EGF were compared to P-11 and C2-9 control cells, respectively. Kidney International 2005 67, 1844-1854DOI: (10.1111/j.1523-1755.2005.00283.x) Copyright © 2005 International Society of Nephrology Terms and Conditions